Dorsal aorta catheterization in rainbow trout (Salmo gairdneri). I. Its validity in the study of blood gonadotropin patterns

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Dorsal aorta catheterization in rainbow trout (Salmo gairdneri). I. Its validity in the study of blood

gonadotropin patterns

Yonathan Zohar

To cite this version:

Yonathan Zohar. Dorsal aorta catheterization in rainbow trout (Salmo gairdneri). I. Its validity

in the study of blood gonadotropin patterns. Reproduction Nutrition Développement, 1980, 20 (6),

pp.1811-1823. �10.1051/rnd:19801007�. �hal-00897783�

Dorsal aorta catheterization in rainbow trout (Salmo gairdneri)

I. Its validity ^{in the} study ^{of blood} gonadotropin patterns

Y. ZOHAR

Laboratoire de Physiologie ^{des Poissons, I. N. R. A.,} Campus Universitaire de Beaulieu, Avenue du Général Leclerc 35042 Rennes Cedex France.

Summary. ^{The use of a dorsal aorta} catheterization technique ^to study gonadotropin

secretion patterns ⁱⁿ the raibow trout was tested. Heparin ^{used to flush the cannula between} repetitive samplings ^{did not have} any ^{effect on} plasma GTH levels. Catheterization resulted in a slight short-term change ^{in those} levels. The gonadotropin levels returned to their initial values as soon as 30 min to 6 hrs after the operation. ^{From then} on, the GTH levels remained close to the initial values in fish exhibiting ^normal feeding behaviour, ^whereas they ^{tended to decrease in « stressed »} females which did not eat normally. ^{The fish which} adapted ^{well to} dorsal aortic catheterization did not show _any changes ^{in the diurnal} pattern ^of GTH levels or in normal gonadal ^{function and GTH} profiles during ^the processes of oocyte maturation and ovulation. It is concluded that individual catheterized trout ^can be used advantageously ^for studying gonadotropin ^secretion patterns ^{after a} 3-day ^reco-

very period and the elimination of those fish which neither resume normal feeding ^nor

return to initial, pre-operative GTH levels. Using ^this technique, ^{it was} demonstrated that

hypophysial GTH release in trout with oocytes undergoing ^active vitellogenesis ^is probably

effected by short-term bursts (pulses) ^{of secretion.}

Introduction.

It is common practice ^to catheterize blood vessels in different mammalian species

in order to follow the metabolic or hormonal profiles. ^When studying ^the temporal patterns ^{of various} reproductive ^{hormones in} the circulation of rainbow trout, ^{it is} desirable ^{to use} individually catheterized fish because these parameters vary widely

and are not synchronized ⁱⁿ different fish of the same population (Crim ^{et at., 1973,}

1975 ; Billard et al., 1978 ; Fostier et al., 1978). ^{Also, it is better to} sample ^{the blood of}

cannulated animals than to do repetitive sampling by ^{heart or} caudal vessel puncture

which may disturb the normal progress of ^a given physiological process and ^cause

mortality. ^{This is true} for any research necessitating frequent sampling ^{of the same}

fish, such as the determination of short-term hormonal fluctuations or rhythms ^or

assessment of the effects of administered substances on various blood ’components.

Some studies ^on teleost species ^have reported ^a vascular catheterization technique

(Smith ^and Bell; 1964 ; Garey, ^{1969 ; Houston, 1971 ; Soivio et al., 1972 ;} Soivio and

Nyholm, 1975). Most of these works as well as others (Houston ^et al., 1969) ^investi- gated changes ^{in a} variety ^of hematological parameters ^or metabolite levels after cannulation. Cannulated fish have only been used once in a study involving ^the

measurement of hypophysial hormone levels (Crim ^{and Cluett,} 1974).

In some mammalian species, ^the plasma levels of several hypophysial ^hormones

have been shown to be significantly ^affected by the method of blood collection (Neill,

1970 ; ^{D6hler et} al., 1977 ; ^{Simms et} al., 1978). ^{The aortic} cannulation of rats resulted in modified patterns ^of prolactin ^and LH secretion during the estrous cycle (Neill, 1972). ^Also, anesthetization and surgery ^were shown to affect hypophysial ^hormone

secretion patterns (Ajika ^et al., 1972 ; Krvlich et at., 1974 ; ^{Glass et} al., 1978 ; Wang

et al., 1978). ^{To our} knowledge, ^no study has considered the possible ^{effects of dorsal}

aortic catheterization (surgery and the presence of ^a cannula in a blood vessel) ^on

the circulating ^levels of any ^{of the} hypophysial ^{hormones in fish.}

As a consequence, ^we attempted ^{to determine if a dorsal aorta} cannulation tech-

nique ^{could be used to} investigate blood GTH patterns ^{in the} free-swimming ^trout (Salmo gairdneri). ^A complementary study (Bry ^{and Zohar,} 1980) ^has investigated

clinical stress indicators to determine how long ^it takes the fish to recover from cathe- ter implantation.

Materials and methods.

Animals.

All the rainbow trout used were 3 to 4 years old and weighed ^{0.8 to 2} kg.

They ^were acclimated to the experimental ^{tanks at least 15} days ^before being ^handled

and were fed once a day between 10 a.m. and noon. The cannulated fish were kept in individual 100-1 rectangular ^{tanks or in 60-1} compartments ⁱⁿ bigger tanks. The water temperature ^was maintained at 10 ! 1 °C between December and May ^and

at 14 ! 1 °C between June and November.

Catheterization surgery. - The catheterization procedures ^{were based on} those pre-

viously ^described (see Introduction), ^{with some} modifications.

The fish were anesthetized in a 300 ppm phenoxy-ethanol solution and placed ^on

the operation ^{table, their} gills perfused ^with refrigerated ^water containing ^{the anes-}

thesia.

A metal syringe ^{needle, with an inner diameter} closely fitting ^{the outer} diameter of the cannula (Clay ^{and Adams} PE-50), ^{was used for aortic} penetration. ^{The 80-cm} long

catheter, filled with a 0.9 p. 100 ^{saline solution} containing ^100-150 unitsiml ^{of sodium} heparin, ^was placed ⁱⁿ the needle which ^was entered at the midline of the buccal roof,

slightly ^{anterior to the first} gill ^{bars, and} cautiously guided posteriorly ^{at about a 30 0} angle. Penetration of the dorsal aorta was indicated by ^blood rushing ^into the cannula :

we then pushed the cannula 8 ^cm up ^into the blood vessel, and gently ^{withdrew the} guiding needle. When penetration ^was successful, there was no hemorrhage. ^The

cannula ^was then attached to the mouth roof, passed through ^{a hole in the snout} slightly ^{anterior to the nasal} opening, and sutured to the snout. Finally, ^{the dead}

space of the cannula ^was filled with an heparin ^{solution, and} the cannula was plugged

with a blocked syringe ^needle adapted ^to a cork floater. It took 8 to 10 min to operate

on one fish.

All blood sampling via the catheter was done after a 200-y l sample, corresponding g to the cannula dead space, had been eliminated. When sampling ^was complete,

the blood filling the cannula was pushed ^back by ²⁰⁰ y l ^of heparinized saline solution.

Experimental procedures for studying :

A. The effect of heparin ^on plasma GTH levels.

At the end of October, ^we selec- ted 16 males whose testes were beginning spermiation ^and 13 females having oocytes

in the ^last stages of vitellogenesis. ^After anesthesia, 500 y l ^{of blood was collected via}

the caudal vessels, and the fish were given ^an intracardiac injection of either 500 U/kg

of heparin ^{in an isotonic} saline solution or of 250 y l/ kg of saline alone. The injected

dose of heparin ^was calculated so as to provide ^{7 to 10 times the amount estimated}

to be in the circulation after ten successive blood samplings ^{via the aortic cannula.}

Blood was collected 4, 8 and 24 hrs after injection. ^The gonadotropin ^{levels in the two} experimental groups ^were compared by ^an analysis ^{of variance. The F-test was used}

for testing ^the linearity of the observed curves and Student’s t-test for comparing ^the

curve slopes.

B. Short and mean-term effects of f cannulation ^on plasma GTH levels.

The experi-

mental protocol ^{of this} experiment has been described in detail by Bry ^{and Zohar} (1980). Briefly, ^{6 fish were} acclimated ^to individual experimental tanks before being operated for dorsal aortic cannula implantation. ^Blood (600 y l ) ^{was withdrawn} (i)

from the caudal vessels of the non-anesthetized fish prior ^{to the} operation (sample

- 10 min) ^and (ii) ^{via the aortic cannula at} the end of the operation ^{about 10 min} later, then 30 min, 6 hrs and 1, ^{3 and 5} days ^after cannulation. The oocytes ^{of one}

of the females (female A) ^{were in the last} stages of vitellogenesis, whereas the other females (B ^to F) had ovulated and their eggs had been stripped ^at least 3 weeks prior

to cannulation. The pattern of GTH levels during ^the experimental period ^{was ana-} lyzed by ^an analysis ^{of variance.}

C. Profiles of plasma GTH levels in cannulated and non-cannulated females.

^The experiment ^{was carried out in} April. A group of 9 females ^was acclimated for one

month to individual tanks exposed ^{to natural} photoperiod. After blood sampling ^via

the caudal vasculature (sample 0), the dorsal aorta was catheterized and the fish

were put ^{back into} their tanks for an additional 5 days. At the end of that time, 300 I !-I

of blood were sampled ^{via the} cannula every 4 hrs for 24 hrs. When the experiment

was terminated, all the fish were killed and their ovaries were examined individually

under ^a dissecting microscope.

Four additional groups of 7 females each ^were acclimated ^to 600-1 tanks exposed

to natural photoperiod. ^After acclimatization, ^{the blood of each} _group ^was sampled

twice (interval ^{of 48 hrs or more between two} samplings ^{of the same} group) ^{at dif-}

ferent times of the day ^{so as to} constitute a sampling pattern parallel ^{to that} of the cannulated fish, ^i.e. every 4 hrs for 24 hrs. Blood (300 y l ) ^was withdrawn from the caudal vessels after rapid 1-min anesthetization. At the end of the experiment, ^the

fish were killed to determine the stage of oocyte development. ^{The two} experimental

groups ^were compared by analysis of variance, whereas the pre and post-operative

GTH levels in cannulated fish were compared by ^{Student’s t-test.}

D. Effect of f cannulation ^{on the} processes of oocyte maturation and ovulation and on

concomitant plasma GTH levels.

Dorsal aortic catheterization was performed ^{on a}

group of 5 females having oocytes ^{in the} migrating germinal ^vesicle stage. ^{The deve-} lopmental stage ^{of the} oocyte ^was determined according ^{to Jalabert et al.} (1976) ^after

a few oocytes, stripped from the females by ^abdominal pressure, had been examined under a dissecting microscope. ^Blood (350 ( J ) ^was sampled from the caudal vessels prior ^to cannulation and via the cannulae every 3 days after cannulation until ovula- tion occurred. The fish ^were anesthetized and stripped ^of a few oocytes ^for microscopic

examination after all post-operative sampling.

A group of 5 non-cannulated females, showing oocytes ^with beginning migration

of the germinal ^{vesicle, was} exposed ^to similar conditions and served as a control.

After anesthetization, the fish in this group ^were bled through the caudal vessels every 3 days ^until they ^{had ovulated. The blood was} always sampled ^{at the same time of} day (10 : ^{00 to 11 : 00} a.m.) ^{in both} experimental groups.

E. Individual daily ^GTH profiles ⁱⁿ cannulated females. ^{-The aim of the} present experiment ^{is to} present ^a study of blood GTH patterns using individual cannulated females, and to determine the criteria to be used for selecting ^reliable animals. Six females acclimated to individual tanks exposed ^{to natural} photoperiod were cannu-

lated in September. ^{Prior to} surgery, 350 yl ^{of blood were} sampled from the caudal vasculature of anesthetized fish within 1 min after netting (sample 0). ^Six days ^after

surgery, 300 y l ^{of blood were} sampled ^via the cannula every hour for ^a 10-hr period

which included the time of the sample 0. Post-mortem examination of the ovaries showed oocytes ^{in active} vitellogenesis ⁱⁿ all females. The presence of peak ^{GTH levels} (outliers) ^{was tested} by Dixion’s gap ^test (cited by ^Bliss, 1967), ^{and the mean of the}

basal (inliers) GTH levels was calculated. Pre-operative ^and peak GTH values were

compared ^{to this mean} using ^{Student’s t-test.}

Gonadotropin radioimmunoassay (RIA).

^{All the} plasma samples ^{were stored}

at― 20 ° C until assayed. ^The gonadotropin ^{levels were measured in} triplicate ^accord- ing ^to Breton and Billard (1977). Within-assay variability (the coefficient of variation of the values obtained for three plasma samples, ^{each run several times in the same} assay) ^{was 0.10} (X

1.39 ng GTH/ml, ⁿ

4) ^{and 0.07} (X

6.53 ng GTH/ml, ⁿ

⁶

and X

_{18.08 ng} GTH/ml, ⁿ

6). ^{The existence of} peak GTH levels in some samples (figs 2, 3) ^{was verified} by assaying ^{the same} samples again ^to eliminate any ^error due to RIA.

In order to test the putative ^{effect of} heparin ^{on the} validity of the RIA results,

a few standard curves were run : in each test, ^a different dose of heparin ^{was added}

to all the tubes. No significant differences ^were found when the parameters ^charac- terizing the different standard curves (slope, intercept, coefficient of regression) ^were statistically compared, ^{and there was no} significant difference between the experimen-

tal and the control ^curves.

Results.

A. Effect of heparin ^on plasma GTH levels (fig. 1).

^{The mean} GTH levels in both

sexes were different in the two experimental groups. ^These divergences ^were present prior ^{to treatment} (sample 0) ^{end were} probably accidental.

The most clear phenomenon ^{in all the} experimental groups ^was the significant

decrease in plasma GTH levels during ^the experiment (analysis of variance ; ^P < 0.01).

The shape of the four curves was not significantly different from linearity (F-test ;

P > 0.05). The estimated slopes ^{of the curves} describing the GTH levels in heparin

and saline-treated fish did not differ significantly from each other in either sex (fig. ^{1 ;}

Student’s t-test ; P > 0.025) ^{and the} curves were therefore nearly parallel. ^Moreover,

a multifactorial analysis ^{of variance} demonstrated that the temporal plasma ^GTH patterns ^{in both} sexes were independent ^of treatment, saline or heparin injection.

Therefore, despite ^the pre-treatment (sample 0) differences in the plasma ^{GTH levels}

of saline and heparin-treated ^fish, neither group ⁱⁿ either sex showed any divergence

in the temporal ^hormone pattern.

B. Short and mean-term effects of cannulation on plasma GTH levels (fig. 2).

^The

GTH levels in six females for 5 days after surgery ^are shown in figure 2. In females A, C, ^{D and} E, ^we observed ^a slight ^but significant ^increase between the initial levels

(sample ^-10 min) and those immediately found after cannulation. Gonadotropin

levels returned to their original values within 30 min to 6 hrs after cannulation. The GTH levels in females B and F remained constant during ^{the same} period. ^{Two dif-}

ferent patterns ^were then evident : in female A (which refused food after cannulation)

and females B, C, ^{E and F} (exhibiting ^normal feeding behaviour), ^{the GTH levels}

remained close to or fluctuated around the initial ones, whereas in female D (refusing

food after cannulation), the GTH levels declined continuously during ^the experimental period.

C. Profiles of plasma GTH levels in cannulated and non-cannulated females (fig. ^3,

table 1).

^An analysis ^{of the} stage ^of oocyte development showed that oocytes ^of

both groups ^{were at} the onset of vitellogenesis. Oocyte ^{diameter in} the cannulated

fish ^was 1 114 ! 135 ym (X:1: SD), ^{whereas in the} non-cannulated trout it was

1 155 ± ¹³⁹ ym. All the females serving ^{in the} present experimeni’ exhibited normal

feeding behavior when blood sampling ^started.

Figure 3 shows the profiles ^of plasma GTH levels in both experimental groups.

During ^{the 24-hr} period, ^mean hormonal levels were slightly, ^{but not} significantly,

lower in the cannulated fish then in the non-cannulated fish. This may reflect ^a GTH level difference between the ^two experimental _groups which existed prior ^{to cannula-}

tion, ^as the GTH level ^mean before cannulation (X o ) ^was lower than or equal ^{to all}

the mean hormonal levels ⁱⁿ the non-cannulated fish (fig. 3). ^{In addition, in none of}

the cannulated fish did the GTH levels differ significantly ^{before or} after cannulation

(table 1). ^The patterns ^{of GTH levels in} both groups during ^the experimental period

were similar, ^{as both curves} closely ^{fitted a} polynomial ^function of the third order.

Mean hormonal levels in both groups tended ^to decline slightly ^{towards noon, then}

to increase during ^the afternoon, reaching maximal levels between 8 p.m. and mid-

night, after which they ^{tended to} decrease again.

D. Effecf of cannulation on the processes of oocyte maturation ^and ovulation and on

concomitant plasma GTH levels (fig. 4).

All the fish exhibited normal feeding ^beha-

viour during ^{the entire} experimental period. The control as well as the cannulated females underwent oocyte maturation and ovulation within the 20 days ^of experimen-

tation. The plasma GTH levels in all females in both the groups increased progressi-

vely during germinal ^vesicle migration ^and maturation to reach maximal levels ^once

ovulation had occurred.

E. Individual daily ^GTH profiles ⁱⁿ cannulated females (fig. 5).

^{Plasma GTH levels}

in vitellogenetic ^females prior ^to cannulation, ^and during ^{a 10-hr} period ⁶ days ^after

cannulation, are ^shown in figure ^5. We observed ^a fluctuating ^{GTH level} profile ⁱⁿ

females G, H, ^{I and} J, ^all exhibiting ^normal feeding behaviour. Peak levels occurred

once or twice during ^the sampling period. ^{Hormonal levels close to the} pre-peak

ones were reached 1 to 2 hrs after the peak ^values appeared. In females K (normal feeding) ^{and L} (refusing food), ^no fluctuations were observed and the GTH levels

were relatively ^{constant. The mean of the basal GTH levels was} calculated for every female after eliminating ^the peak (outliers) ^values (Dixion’s gap test ; fig. 5). ^{In none}

of the females G to K was the pre-cannulation plasma ^{GTH level} significantly ^diffe-

rent from the mean of the basal levels calculated for the ^same females (Dixion’s

gap test, Student’s ^{t-test :} P > 0.05). ^{In female L, the} only ^one refusing ^{food, the GTH}

level before cannulation was significantly higher ^{than the mean} of the basal values

(all ^{values in this} case) ^{in that fish once it was} cannulated and serially sampled.

Discussion.

The blood sampling ^of unanesthetized, free-swimming ^{fish is} advantageous ⁱⁿ studying ^certain physiological functions. However, dorsal aortic catheterization involves a traumatic surgical intervention and a long-term implanted ^{cannula in the}

blood vessel, both of which may disturb the normal profile ^{of a} given physiological

process and induce ^{a transient or} chronic stress situation. Repetitive ^blood sampling

via an implanted cannula may have the ^same effects, ^as it causes changes ^{in blood}

volume and the possible introduction of a certain amount of heparin ^into the circula- tion. Therefore, ^when cannulating ^{fish, the} advantages and limitations of the technique

must be considered ⁱⁿ relation to the function ^to be studied.

During ^the operation for catheter implantation, ^{the fish are} exposed ^{to several} stressing ^{factors, i.e.} anesthetization, _surgery ^and hypoxia : ^{the first two have both}

been found to influence plasma gonadotropin ^{levels in} different mammalian species (see Introduction). ^{In the} present study, ^{there was a} slight rapid increase in GTH levels after cannulation in 4 of the operated fish. That change ^was concomitant with an impor-

tant increase in the hematocrit and the glucocorticoid ^{levels in the same females}

(Bry ^{and Zohar,} 1980), ^and might reflect either hemoconcentration and/or ^a possible

relation between a stress situation and the hypophysial secretory mechanims. The

two different patterns of GTH level response (increased ^or steady levels), ^observed

in the different fish may represent individual variations in the temporal pattern ^{of the}

same phenomenon, ^{as a} biphasic LH response ^{to acute} anesthetization stress, consisting

of an initial stimulatory phase ^{and an} ensuing inhibitory ^{one, was observed in the}

rat (Krüliche et 01., 1974).

Recovery ^to pre-operative ^{GTH level was} complete ^{within 30 min to} 6 hrs after surgery. Two females, ^{A and D} (fig. 2), ^did not return to normal feeding ^{after cannu-}

lation ; ^{this was} probably ^{due to chronic stress as} implied by the abnormal glucocorti-

coid profiles ⁱⁿ those females during ^{the entire} period (Bry ^{and Zohar,} 1980). ^{In one} (female A), ^{we could not detect any abnormal GTH} profile, whereas the GTH levels decreased progressively ⁱⁿ the other female (D). ^{A similar} phenomenon ^{was observed}

in female L (also refusing ^{to eat after} cannulation), ^{i.e. the} pre-operative ^{GTH level}

was higher ^than all the levels observed ⁶ days ^later (fig. 5). ^{Thus, a decline in GTH}

level ^was found in two out of three « stressed >> females. That decline could have been induced by ^{chronic stress either} directly ^or indirectly ^{as a result of food} deprivation,

as starving ^decreases gonadal activity ^and hypophysial ^and plasma GTH levels in the goldfish (Clemens ^and Reed, 1967 ; ^{Gillet et} al., 1980). In all cannulated females which resumed normal feeding, ^the plasma ^GTH levels observed in repetitive samples

at 6 post-operative days ^{did not} differ from the pre-operative ^{levels. Thus,} the resump- tion normal feeding behaviour and the return to initial gonadotropin levels may be used as routine criteria to test the state of recovery of cannulated fish and the validity

of their use for studying gonadotropin ^secretion patterns. ^The present ^{work as well}

as that by Bry ^{and Zohar} (1980), describing ^the development of glucocorticoid ^levels,

hematocrit, leucocrit and feeding behaviour after cannulation, suggest ^that minimally

stressed fish, adapted ^{to a cannula, can} be available as soon as 3 days after the opera- tion.

Aortic cannulation ^was shown to modify ^blood prolactin and LH levels in rats,

although ^the presence of ^{such a} cyclic phenomenon ^{as the} proestrous secretory surge of both hormones was undisturbed (Neill, 1972). One of the possible ^{reasons for such} changes ^is the presence of heparin ⁱⁿ the circulation. This anticoagulant ^{was shown}

to interfere with LH binding ^to gonadal receptors (Salomon ^and Amsterdam, 1977 ;

Fox and Wisner, 1979). Nevertheless, our study demonstrates an absence of exogenous heparin ^{effect on} plasma ^{GTH levels in male and female trout.} The decline in gonado- tropin ^{levels in} heparin and saline-treated fish was independent ^{of the treatment and} probably resulted from the handling stress to which the fish were exposed during ^the

entire experimental period. ^{Moreover, the} pattern of GTH level for 24 hrs, in fish

sampled ^{via a cannula, was} parallel ^to that observed ⁱⁿ non-cannulated fish blood

sampled by caudal vessel puncture. The cannulation of females with oocytes beginning germinal ^vesicle migration ^{did not} affect the normal continuation of oocyte ^maturation

and ovulation ; gonadotropin ^levels accompanying these processes ^{were not} modified in cannulated animals and exhibited the typical previously ^{described increase} (Jala-

bert et al., 1976 ; ^{Fostier et} ot., 1978). ^Thus, cannulated female trout, well adapted ^to

the cannula, showed gonadotropin ^secretion patterns ^and gonadal activities similar

to those seen in non-cannulated females.

Some of the experiments ^{in the} present study confirm the individual heterogeneity

of plasma GTH levels in fish representing ^{the same ovarian} stage. ^{This fact necessi-}

tates the use of the cannulation technique ^{for the} study ^of plasma gonadotropin patterns. ^An example ^{of the} importance ^{of this} technique ^{is included in} figure ^5.

When cannulated females having oocytes ^{in active} vitellogenesis ^{were bled} hourly

for 10 hrs to determine the gonadotropin ^levels, very different patterns were seen.

The GTH levels in most « unstressed » females showed short-term fluctuations, ^whe-

reas they ^{remained constant in one} female. The pattern ^of plasma ^{GTH level was}

not synchronized ⁱⁿ the different females. These observations suggest ^a possible pulsatile ^{secretion of} gonadotropin ⁱⁿ females with oocytes in active vitellogenesis.

Hontela and Peter (1978) reported significant daily fluctuations in the serum GTH levels in female goldfish undergoing ^ovarian recrudescence. In their study, ^a group of fish was bled and killed at different times so as to constitute a 24-hr period ^{in which} sampling ^{was effected} every 4 hrs ; their results suggest ^{a relative} synchronization

in GTH secretion events among the different females. The ^trout model, ^as shown in

our study, might be different as the individual GTH profiles ^{were not} synchronized, indicating that short-term GTH secretion events were distributed distinctively during

the day according ^to the female. When individual female trout undergoing ^vitello- genesis ^{were bled} (via cannulae) every ^{4 hrs} for 32 hrs, daily fluctuations were

observed in some, but again ^no profile synchronization ^{was found} (Zohar, unpublished data), ^thus confirming the above hypothesis. Other GTH secretion profiles ^were

observed in cannulated female trout ^at different stages ^{of ovarian} development (Zohar, unpublished data).

In conclusion, unstressed, cannulated fish must be used to study the short-term tem-

poral profiles of any of the circulating ^hormones when those profiles, ^{as in the case}

of female trout GTH patterns, ^{are not} synchronized. ^The present study suggests ^a

short-term transient effect of acute surgical ^{stress on} plasma GTH levels in most cannulated fish, ^{and a} long-term effect of surgery ^or the cannula in non-adapted spe- cimens. Nevertheless, following ^a recovery period ^{of at least 3} days and after elimi- nation of chronically ^{stressed females,} individual cannulated fish can be advantageous

models for studying gonadotropin ^secretion patterns ^{as reflected} by the blood hor-

mone levels.

Re 5

u ^en f6vrier ¹⁹⁸⁰ Accepte ^{en mai 1980.}

Acknowledgements.

^{I wish to} thank Miss Aline Solari for aid in statistical analysis,

Mrs. Alice Daifuku for improving ^the English manuscript and Mrs. Denise Bouix for typing ^{it. I am indebted to R. Billard, B. Breton, C.} Bry, ^{A. Fostier} and CI. Weil for fruitful discussions and for critical reading ^{of the} manuscript. The author was suppor- ted by ^a fellowship from the Hebrew University of Jerusalem, Israel.

Résumé. L’utilisation ^{de la} technique ^de cathétérisation de l’aorte dorsale _pour l’étude des profils de sécrétion gonadotrope ^{chez la truite} arc-en-ciel a été testée. L’héparine

utilisée pour rincer la cannule lors des différents prélèvements ^{n’a aucun} effet sur les niveaux

plasmatiques ^{de GTH. La} cathétérisation entraîne une variation faible et de courte durée

de ces niveaux. Les niveaux gonadotropes reviennent à leurs valeurs initiales 30 min à

6 h après l’opération. ^Les concentrations de GTH demeurent alors, proches de leurs valeurs initiales chez les poissons présentant ^un comportement alimentaire normal ; cependant,

chez les femelles « stressées » ne se nourrissant _pas normalement, ^{ces niveaux} ont tendance à diminuer.

Chez les poissons ^bien adaptés, ^la cathétérisation de l’aorte dorsale n’a d’effet ni sur

le profil diurne des niveaux plasmatiques ^{de GTH ni sur} la fonction gonadique ^{et le} profil

de GTH pendant ^le processus de maturation ovocytaire ^et d’ovulation. En conclusion, ^des

truites cathétérisées peuvent être utilisées avec avantage pour étudier les profils ^{de sécré-}

tion gonadotrope, ^ceci après ^un délai de trois jours ^de récupération ^{et en} éliminant les

poissons qui ^ne retrouvent ni leur comportement alimentaire normal ni leurs niveaux initiaux de GTH pré-opératoire. L’utilisation de cette technique ^{nous a} permis ^{de montrer}

que, chez la truite ^en vitellogenèse active, la secrétion de GTH hypophysaire ^s’effectue probablement ^{sous forme de} pulses ^{de courte durée.}

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